1. Field of the Invention
The present invention relates generally to novel phosphotetrahydropyran compounds, primarily derivatives of mannose-6-phosphate, and their use in treating diseases or disorders that are mediated at least in part by T lymphocyte emigration from blood to tissues. In particular, the present invention relates to the use of these compounds and pharmaceutical compositions comprising them to treat T lymphocyte mediated inflammatory and autoimmune diseases in animals and man.
2. Description of the Background Art
The adaptive immune response of mammals may be viewed as being divided into two arms: antibody (or humoral) and cell-mediated immune responses. Different classes of lymphocytes play key role in these two type of responses. Antibody responses are generated by antibody-producing B lymphocytes (or B cells) which differentiate into plasma cells, while cell-mediated immune responses are mediated by T cells, such as cytotoxic T lymphocytes (CTL) which specifically recognize and kill antigen-bearing target cells, such as infected cells or tumor cells. These “effector” T cells commonly recognized their target antigens in the context of major histocompatibility complex (MHC) proteins, usually MHC class I proteins. Both classes of immune responses usually depend upon the action of another set of T lymphocytes, T helper cells, which also recognize antigenic epitopes presented in the context of major histocompatibility complex (MHC) proteins, usually MHC class II proteins. The processes involved in the generation and manifestation of these responses and the roles played by the various classes of lymphocytes in infection are well understood. For a more detailed explication of the foregoing and other description in this section, see immunology textbooks such as Abbas, A K et al., eds., Cellular and Molecular Immunology (4th Ed.), W.B. Saunders Co., Philadelphia, 2000, Janeway, C A et al., eds., Immunobiology, 5th ed., Garland Publishing Co., New York, 2001; Roitt, I et al., eds, Immunology, 5th ed., C.V. Mosby Co., St. Louis, Mo. (2001); Klein, J et al., Immunology, 2nd edition, Blackwell Scientific Publications, Inc., Cambridge, Mass., (1997).
T cells are believed to engage in a process termed “immunological surveillance” which they execute by continuously circulating (recirculating) throughout the body. Recirculation involves migration of T cells from lymph nodes (LN) into the blood stream via the efferent lymphatic ducts and then re-entry into LN from the blood via post capillary venules. T cells also exit the circulation by crossing capillary walls and entering tissues, moving through the tissues, and entering afferent lymphatic vessels draining these tissues, and finally making their way via these lymphatics to local draining LNs which are positioned around the body.
If, during this sojourn through the tissues, T cells encounter an antigen that they recognize specifically, via their clonally expressed T cell receptors (TCR), and to which these cells are programmed to respond, the T cells are activated, leading to a state of cell-mediated immunity. Thus, when recognizing and responding to an infectious agent or other foreign antigen, T cells generate responses that ultimately result in destruction and clearance of the pathogen. In some cases, however, the T cell response may not be controlled optimally and therefore become excessive, resulting in collateral damage to normal tissues in the vicinity of the infectious (or other foreign) agent. In other cases, T cells initiate an inappropriate immune response directed to normal tissue components or “self-antigens.” Irrespective of the mechanism of these “normal,” aberrant or dysregulated responses, when they become clinically apparent, the resulting disease or disorder is often termed an “autoimmune disease.” The cell and tissue damage is commonly referred to as “immunopathology.” Many pathological disorders of humans have been attributed to autoreactive T lymphocytes and the inflammatory responses they induce. See, Gallin, J et al. (eds), Inflammation: Basic Principles and Clinical Correlates, 3rd Edition, Lippincott Williams & Wilkins, 1999. Included-among these immunopathological maladies a number of well-known autoimmune diseases (see, for example, A. N. Theofilopoulos et al. (eds), 2nd edition, The Molecular Pathology of Autoimmune Diseases, Taylor & Francis, 2002)). Examples of these are multiple sclerosis (MS), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), acute disseminated encephalomyelitis (ADE) and insulin-dependent diabetes mellitus (IDDM, also Type I diabetes). Psoriasis too is a T cell-mediated inflammatory disease of the skin (Bos, J D et al., Immunol. Today 20:40-46 (1999)).
Approaches and agents for treatment or prevention of immunopathology and autoimmune diseases, developed over decades, target many and varied facets of the immune and inflammatory processes described above. Though some agents are specific to particular antigens, the vast majority have been nonspecific (see textbook references, supra). Although current approaches have met with varying degrees of success, many carry with them multiple undesirable side effects and risks.
Several investigators have targeted various steps in the T cell migration/extravasation process as an approach to suppressing some of the autoimmune disorders noted above. Several studies by Israeli investigators are described first. Naparstek, Y et al., Nature 310:241-244 (1984) discussed earlier studies of lines of activated T lymphocytes specifically sensitized to the central nervous system (CNS) antigen, myelin basic protein (MBP); upon intravenous inoculation into syngeneic rats, these T cells penetrated blood vessels, accumulated in the CNS parenchyma and caused the inflammatory/immune sequelae manifested as experimental autoimmune encephalomyelitis (EAE), a well-recognized animal model of human MS. These authors studied interactions of activated anti-MBP T lymphocytes with the basement membrane-like extracellular matrix (ECM) produced by vascular endothelial cells. They found that activated, but not resting T lymphocytes, produced an endoglycosidase (heparanase) enzyme capable of degrading heparan sulfate side chains of the proteoglycan scaffold of the ECM and responded to MBP presented by the ECM by enhanced elaboration of this enzyme. These results suggested that tissue-specific antigens on blood vessel walls could direct lymphocyte “homing” by activating enzymes that facilitate penetration of the subendothelial basal lamina. Following up the above study, Lider, O et al., J. Clin. Invest. 83:752-756 (1989), found that administration of low dose heparin to mice inhibited lymphocyte traffic and delayed-type hypersensitivity (DTH) reactions (‘classic’ cell-mediated immune responses). Treatment with commercial or chemically modified heparins at relatively low doses once daily (e.g., 5 μg/mouse; 20 μg/rat) led to inhibition of allograft rejection and two experimental auto-immune diseases (EAE and adjuvant arthritis). The ability of chemically modified heparins to inhibit the migration stages of the immune reaction was associated with their ability to inhibit expression of T lymphocyte heparanase. Importantly, there was no relationship of this T cell inhibitory effect with the heparins' anticoagulant activity. Thus appropriate doses of heparins, even if devoid of anticoagulant activity, could effectively regulate or inhibit undesired T cell migration involved in autoimmune diseases.
Subsequently, Lider, O et al., Eur. J. Immunol. 20:493-499 (1990), reported studies of the effects in vitro and in vivo of the heparanase inhibitor, heparin, on the expression of T lymphocyte heparanase and on the ability of T lymphocytes to mediate a DTH reaction. T cell heparanase activity could be induced in vivo by immunizing mice with an antigen or in vitro by activating T lymphocytes polyclonally with a mitogen. Again, low doses of heparin inhibited the expression of heparanase induced either way. The same doses of heparin that inhibited expression of heparanase also inhibited the ability of LN T cells to migrate to a site of antigen and adoptively produce a DTH reaction. These findings further supported the notion of modulating cell-mediated immunity using heparin which would inhibit expression of T lymphocyte heparanase expression and cell migration. Vlodavsky, I et al. (Invas. Metas. 12:112-127 (1992), further discussed the importance of heparanase in the interactions of T lymphocytes (as well as B lymphocytes, platelets, granulocytes, macrophages and mast cells) with the subendothelial ECM, due to degradation of heparan sulfate by this enzyme. The enzyme is released from intracellular compartments (i.e., lysosomes, specific granules) in response to various activation signals (e.g., antigens, mitogens), explaining heparanase's role in inflammation and cellular immunity. Of interest was the fact that various tumor cells expressed and secreted heparanase in a constitutive manner, which was correlated with their metastatic potential. Thus, utilizing a shared mechanism, T cells and other normal leukocytic cells on the one hand, and metastatic tumor cells on the other, which enter the bloodstream, can travel to distant sites and extravasate to the tissue parenchyma there by means of this cellular heparanase enzyme.
There is clearly a need in the art for new inhibitors of undesired cellular migration, particularly T cell migration, that can be exploited in the treatment of various diseases or disorders associated with inflammation and immune responses that involve such cellular migration as a step in the pathophysiology.
As is described below, a cell surface receptor for mannose-6-phosphate (M6P) on T lymphocytes appears to play a role in their extravasation in vivo. The background to that observation is as follows. Recirculating lymphocytes initiate extravasation from the blood stream by binding to specialized high endothelial venules (HEV) within peripheral LNs and other secondary lymphoid organs. Stoolman, L M et al. (J. Cell Biol. 99:1535-1540 (1984)) reported selective inhibition of lymphocyte attachment to HEV by M6P and related carbohydrates. Yednock, T A et al. (J. Cell Biol. 104: 713-723, 725-731 (1987)) employed a cell-surface probe—fluorescent beads derivatized with ‘PPME”, an M6P-rich polysaccharide—to directly identify a carbohydrate-binding receptor on lymphocytes surfaces. Lymphocyte attachment to PPME beads mimicked the interaction of lymphocytes with LN HEV: both interactions were selectively inhibited by the same panel of structurally related carbohydrates, were calcium-dependent, and were sensitive to mild trypsin treatment of lymphocytes. Thymocytes (and certain thymic lymphoma cell lines) which bind very weakly to HEV, also bound poorly to PPME beads. The authors concluded that a carbohydrate-binding receptor on lymphocytes, detected by these PPME beads, is involved in lymphocyte attachment to LN HEV.
The initiation of lymphocyte extravasation employs a family of cell adhesion molecules called homing receptors that mediate lymphocyte attachment to HEV within the lymphatic tissues. A putative homing receptor was identified by the monoclonal antibody (mAb), MEL-14, which recognized an 80-90 kDa glycoprotein on the surface of mouse lymphocytes and blocked their attachment to LN HEV. The authors examined the relationship between the carbohydrate-binding receptor and the putative homing receptor identified by MEL-14 and found that: MEL-14 completely and selectively blocked the activity of the lymphocyte carbohydrate-binding receptor; the ability of six lymphoma cell lines to bind PPME beads correlated with cell-surface expression of the MEL-14 antigen, as well as LN HEV-binding activity; selection of lymphoma variants that bind to PPME-beads produced highly correlated and selective changes in MEL-14 antigen expression. The authors concluded that the carbohydrate-binding receptor on lymphocytes and the MEL-14 antigen, which have been independently implicated as receptors involved in LN-specific HEV attachment, are very closely related, if not identical, molecules.
A group of investigators in Canberra, Australia, that included one of the present inventors (Cowden) studied the ability of phosphosugars to inhibit CNS inflammation (Willenborg, D O et al., FASEB J. 3:1968-1971 (1989)). They found that adoptively transferred EAE was inhibited by various phosphosugars, particularly M6P. The authors speculated that the sugar specificity may be due to depletion of lymphocyte cell-surface lysosomal enzymes that are essential for the passage of lymphocytes across the vascular endothelium and entry into the CNS parenchyma. A later study by the same group (Willenborg et al. Immunol. Cell Biol. 70:369-377 (1992)) showed that development of joint inflammation in a model of adoptively transferred arthritis in rats was also inhibited by treatment with M6P and by the alkaloid inhibitor of α-glucosidase, castanospermine (CS). M6P was effective at a dose of 25 mg/kg per day delivered via mini-osmotic pumps implanted either subcutaneously (sc) or intraperitoneally. CS was given orally in the drinking water (actual dose ˜60-65 mg/kg per day), which treatment greatly reduced inflammatory infiltrates in the synovium and surrounding tissue. CS also inhibited disease progression when treatment was commenced after the onset of symptoms. The authors speculated that the mechanism(s) of action included inhibition of the passage of leucocytes through vascular subendothelial basement membranes by inhibiting the function or expression of leucocyte cell surface-bound enzymes that are essential for such migration.
In another study by the same group (Bartlett, M R et al., Immunol. Cell Biol. 72:367-374 (1994)), M6P, CS and some sulfated polysaccharides (SPS) were tested in murine models of allograft rejection and elicitation of peritoneal exudates. CS, M6P and the SPS, fucoidin (or fucoidin), partially inhibited rejection of permanently accepted thyroid allografts (induced by the i.p. injection of donor strain allogeneic spleen cells). Elicitation of inflammatory exudates by thioglycollate was inhibited by CS, M6P and fucoidin with sustained leukopenia being induced by CS. In contrast, CS and fucoidin, but not M6P, inhibited antigen-elicited peritoneal exudates. The authors claimed that, while these results suggested that CS, M6P and the fucoidin exhibited subtle differences in their anti-inflammatory activity, the mechanism of inhibition was at the level of leukocyte extravasation.
The Canberra group directly tested the hypothesis that heparin, M6P and CS mediate their anti-inflammatory effects by inhibiting the passage of leukocytes through the subendothelial basement membrane (SBM) (Bartlett et al., J. Leukoc. Biol. 57:207-213 (1995)). These three compounds were examined for their ability to prevent the in vitro degradation of a 35SO4-labeled ECM by neutrophils, lymphocytes, endothelial cells (ECs), and platelets. While all three compounds inhibited ECM degradation, M6P and CS were cell-type specific in their effects. Heparin inhibited the heparanase activity of all cell types examined, confirming the results of previous studies (discussed above). M6P selectively inhibited lymphocyte heparanase but not that of platelets, neutrophils, or ECs. CS selectively inhibited induced EC heparanase and sulfatase activity but did not affect the constitutive expression of these degradative enzymes by unstimulated ECs. The results were said to support the view that leukocytes markedly differ in the mechanisms by which they degrade SBM/ECM to enable extravasation.
In a review article (Parish, C R et al. Immunol. Cell Biol. 76(1):104-13 (1998)), the Canberra group discussed the inadequacy of current anti-inflammatory drugs in the treatment of MS and other inflammatory diseases because (a) disease progression was not arrested and (b) undesirable side effects posed problems. They discussed their decade-long (see studies described above) development of novel drugs that could interfere with the entry of leucocytes into inflammatory sites by inhibiting their passage through the SBM. An important point emerging from their research was that breach of the SBM is a cooperative process, involving activation-induced and cytokine-induced degradative enzymes contributed by leucocytes, endothelial cells and platelets. This document described the properties of three separate classes of anti-inflammatory compounds: (1) phosphosugars, (2) sulfated polysaccharides/oligosaccharides and (3) CS, all of which inhibit the passage of leukocytes through SBM. Each “drug” type appears to prevent SBM degradation by a different mechanism. Sulfated polysaccharides/oligosaccharides mediate their anti-inflammatory effect by inhibiting the endoglycosidase, heparanase, which plays a key role in the solubilization of SBM by invading leucocytes. Phosphosugars probably inhibit inflammation by displacing lysosomal enzymes involved in SBM degradation from cell surface M6P receptors. This mechanism—expression of degradative enzymes on the cell surface—was particularly evident in activated T lymphocytes. For reasons which were said to be unclear, CS specifically inhibits SBM degradation by ECs, which results in a characteristic perivascular arrest of leucocytes in inflammatory sites. The review concluded that inhibitors of SBM degradation represent viable anti-inflammatory agents for future development.
A more recent publication by the Canberra group (Hindmarsh, E J et al., Immunol Cell Biol 79:436-43 (2001)) evaluated the antiinflammatory action of M6P, notably in the inhibition of EAE and adjuvant-induced arthritis in rats. It was proposed that M6P exerted its anti-inflammatory effect by displacing lysosomal enzymes (which are involved in T cell extravasation into inflammatory sites) from the 300 kDa M6P receptor (=MPR-300) on the T cell surface. The authors hypothesized that MPR-300 would be selectively expressed on the surface of activated T cells, as T cell entry into the CNS in EAE depends on the activated state of the cells. They therefore examined (a) correlation between cell surface expression of MPR-300 on T cells and their state of activation, and (b) whether T cells in inflammatory sites expressed the receptor. Flow cytometric studies showed MPR-300 was absent from the surface of unstimulated rat T cells isolated from peripheral blood and lymphoid tissues, and from T cells resident within the peritoneal cavity. In contrast, MPR-300 was expressed on activated T cells derived from an inflammatory peritoneal exudate. In vitro studies demonstrated transient expression of MPR-300 on the surface of splenic T cells following stimulation with Con A. MPR-300 was also induced on T cell lines by antigen stimulation. The authors concluded that T cells in inflammatory sites express MPR-300 on their surface and that activation of these cells induces cell surface expression of this receptor. Such findings were said to be consistent with the notion that cell surface MPR-300 is required for the entry of T cells into inflammatory sites.
A commonly owned PCT application published as WO/0204472, exploited the foregoing observations by the Canberra group and described various novel M6P derivative compounds and their use in treating diseases that are dependent upon T lymphocyte migration.
As a next step in the development of effective inhibitors of T cell migration and extravasation, the present inventors have discovered yet other, improved phosphotetrahydropyran compounds (distinct from those in WO/0204472) that are defined by Formula I, below. The compounds of this invention are more resistant to endogenous mannosidase and phosphatase enzymes, are effective inhibitors of T lymphocytes migration from the blood into tissues and are thus useful additions to our armamentarium of treatments for autoimmune diseases and, in general, for any disease or disorder that involves such T lymphocyte migration in its pathogenesis.